Demyelination and Multiple Sclerosis.
Demyelination is a neuropathological state where the insulating myelin sheath on the axons of the neurons is degraded, the pathogenesis of which could be due to a variety of causes.1 Multiple sclerosis (MS), one such clinical condition, is a chronic and most common demyelinating disease, affecting about 2.2 million people worldwide.2 It is characterized by a patchy degradation of myelin on the axons, known as demyelinated lesions, and the healing of these patches occurs via scar formation called plaques.
A variety of causes such as genetic, immunological and environmental factors are suggested to play a role leading to this condition.3 The most common theory is the autoimmune theory which postulates that sensitization of T cells in the periphery leads to their travel through a disrupted blood brain barrier to attack and destroy myelin.4 Several CNS proteins have been shown to induce this condition, including myelin proteolipid protein (PLP),5 myelin-associated glycoprotein (MAG),6 myelin oligodendrocyte glycoprotein (MOG),7 transaldolase and S100.6 Genetic studies indicate the involvement of about 30 single-nucleotide polymorphisms (SNPs), although it remains to be seen as to the relevance of these SNPs for MS therapeutics development.
Current MS therapies reduce the frequency of relapses but do not delay the progression of the disease nor do they reverse the destruction of myelin.9,10,11 The most popular treatment is Copaxone™ (also known as Copolymer-1, Cop-1, or Glatiramer acetate), marketed by Teva Pharmaceuticals. This is an immunomodulator drug and is a random polymer of four amino acids, glutamic acid, lysine, alanine and tyrosine in the same proportion found in myelin basic protein (MBP).
The mechanism by which Copaxone™ exerts its effects in MS patients is not completely understood. However, it is believed to act by modifying immune processes that may be responsible for the pathogenesis of MS. Studies in vitro and in vivo suggest that upon administration, Copaxone™-specific suppressor T cells are induced and activated in the periphery.12,13 There are several side effects associated with Copaxone™, and this drug is not completely effective in delaying the onset of severe or fulminating MS.11 
Another popular drug is cannabis extract (dronabinol) used by MS patients due to its pain relief effects. A clinical study in the UK (the CUPID study) to determine the ability of dronabinol to slow disease progression in primary progressive and secondary progressive MS is currently underway.
Other clinical trials, for example, involving fingolimod (Gilenya™ by Novartis) to test safety and effectiveness of this drug in primary progressive MS, laquinimod vs. interferon β-1a (Avonex®) vs placebo to assess the compound in relapsing-remitting MS and teriflunomide (HMR1726) to assess the compound in clinically isolated syndrome (CIS), for relapsing-remitting MS, are actively being pursued in various late stages.11 
A much anticipated oral therapy cladribine (Movectro™) for the treatment of relapsing forms of multiple sclerosis has been recently withdrawn from clinical trials by Merck Serono due to its inability to meet U.S. FDA requirements.14,15 Cladribine—an immunomodulator—was believed to work by interfering with the activity of white blood cells in the central nervous system, thereby interrupting the immune attacks that cause the unpredictable symptoms of MS. It must be noted that cladribine in injectable form is used to treat hairy cell leukemia, thus raising severe safety concerns for long term use in MS patients simply based on its molecular mechanisms of action.
Currently, there is a desperate need for novel mechanisms of preventing and potentially reversing demyelination, such that the treatment options for demyelinating diseases such as multiple sclerosis can be conceived with better safety profiles and with clear molecular mechanisms of action.
Citrullination and Demyelination.
In general, immunological self-tolerance is an important defense against many autoimmune diseases and its breakdown in the body leads to various autoimmune diseases. This primarily arises from the immune recognition of self-proteins that have undergone post-translational modifications under pathophysiological conditions that would not happen under normal circumstances.
Citrullination, a post-translational event, in general is involved in many cellular processes such as gene regulation, embryonic development and differentiation.16,17 Lately, the abnormal role of (hyper)citrullination in a variety of diseases has been uncovered, including in MS, rheumatoid arthritis, Alzhelmer's, scrapie, psoriasis and Creutzfeld-Jacob disease.18,19 Thus the generation, metabolism and regulation of citrullinated proteins have become a major focus of research.20,21 For example, deimination (or citrulination) of histone H3 is correlated to apoptosis of human neural stem cells, and inhibition of citrullination showed reduced apoptosis and less tissue loss as well as enhanced regeneration of neural cells.17 
In MS, extensive studies of hypercitrullinated MBP indicated that MBP, a key component of the myelin sheath and critical for the maintenance of myelin compaction, contained the non-coded amino acid citrulline in abnormal proportions. In normal brain, the “citrullinated MBP” accounts for 20% of the total MBP, whereas in chronic MS it accounts for 45%22 and in fulminating MS it is 90% of the MBP.23 In a number of studies using a variety of biophysical techniques,24,25,26,27,28 it was demonstrated that citrullinated MBP prevented compaction of the bilayer, resulting in destabilization of the membrane and subsequent degradation leading to demyelination, and an irreversible damage to the axons.29,30 
Thus, hypercitrullination is at the root of neuropathogenesis due to demyelination. In the central nervous system, peptidyl arginine deiminases (specifically PAD2 and PAD4) are responsible for the citrullination.
PAD Enzymes and Citrullination.
Peptidyl arginine deiminase (PAD) catalyzes the post-translational citrullination of proteins.31, 32, 33 Citrullination is the process of deimination of Arg residues on select proteins, or in other words, transformation of Arg into citruline via deimination (Scheme 1). There are five isozymes of PAD that exist in humans: PAD-1, -2, -3, -4 and -6. Their expression in tissues varies significantly, regulated by transcriptional and post-transcriptional mechanisms. PAD2 and PAD4 are specifically implied in multiple sclerosis, as enhanced levels of these two isoforms are observed in CNS under inflamed conditions.21,34,35

There is convincing evidence in vivo that higher levels of PAD activities and hypercitrullination are observed in MS.36 For example, a routinely used MOG-EAE model for MS, which is a CD4(+) T cell-driven model, induced with the immunodominant 35-55 peptide of myelin oligodendrocyte glycoprotein (pMOG35-55) was used to test whether citrullination of a T cell epitope can contribute to disease etiopathology.29,37 In this experimental model, the PAD2 and PAD4 enzymes were significantly upregulated in the inflamed CNS of the animals. T cells that responded specifically to the citrullinated pMOG could not initiate the EAE lesion, but these cells could provoke exacerbation of pathology if transferred into mice with an ongoing EAE. This experiment strongly suggested that once inflammation in MS is established, citrullination of target autoantigens can allow an expanded repertoire of T cells to contribute to CNS pathology, and enhanced levels of PAD enzymes are observed in these tissues.37 A similar study using the peptides from myelin basic protein (MBP) epitopes indicated that self-antigens could potentially trigger the disease in susceptible individuals carrying citrullinated peptide epitopes.38,39 
In an elegant study by Oguz et al., it was shown that citrulline is more frequently identified in the brains of patients in vitro with an early onset of the MS disease than in the healthy subjects using magnetic resonance spectroscopy.40 This study and others established the direct correlation between hypercitrullination and the disease progression in MS.41 
Raijmakers et al. reported that PAD2 knockout mice developed EAE despite the lack of PAD2 which suggested hypercitrullination may be irrelevant in MS.42 However, the Moscarello group collaborated and obtained these PAD2 knockout mice from Raijmakers' lab, extracted MBP from whole brain digested with trypsin and resolved the peptides by mass spectrometry. Several citrulline-containing MBP peptides were discovered and confirmed that citrullinated MBP was present in the PAD2 knockout mice.44 In addition, citrullinated CNPase (cyclic nucleotide phospohydrolase, a myelin enzyme) and MOG (myelin oligodendrocyte glycoprotein) were also detected in these samples. The citrullinated MBP was generated by PAD4 that is present in the brain and spinal cord (as does PAD2, if it were present). The PAD2 knockout mice contained similar amounts of PAD4 as the wild type mice.44 In summary, protein citrullination is an active process in the PAD2 knockout mice due to PAD4 activity. Thus, investigations on deiminases and the inhibition of PAD2 and PAD4 enzyme activities are important challenges in pursuit of understanding demyelinating diseases.21,43,44,45 
Inhibitors of PAD Enzymes:
A non-specific, active site PAD inhibitor, 2-chloroacetamidine (2CA), attenuated MS disease, decreased the amount of citrullinated protein and decreased PAD activity in the brain in four animal models of MS: two neurodegenerative and two autoimmune disease models.46 
Protein citrullination, expression of PAD protein and the corresponding enzyme activity in extracts of normal and of normal-appearing white matter (NAWM) from MS patients have been investigated (FIG. 1).47 PAD2 protein expression was elevated in NAWM from MS brain, with a corresponding increase of PAD activity and protein citrullination (FIG. 1A).48 Since PAD4 translocates into the nucleus and affects the transcription, it was of interest to look at PAD2 mRNA levels after treatment with 2CA. A decrease in the mRNA levels indicates the potential inhibitory effect of 2CA on PAD4 activity. The elevated activity of PAD in normal-appearing white matter is 2-4 fold that in the normal tissue. These levels of PAD also correlate well with the elevated levels of citrullinated protein in the white matter of the MS patients, in comparison to that in normal brain white matter (FIG. 1B). Following the addition of 2CA to NAWM extracts, PAD activity declined, demonstrating that 2CA was effective in human brain extracts dampening the enzymic activity of PAD (FIG. 1C), although 2CA is a non-specific inhibitor. 2CA targets all PAD isozymes since it's a non-specific inhibitor.
2CA is a covalent inhibitor of PAD4 (FIG. 2). This inhibition pattern was confirmed by treating PAD2 or PAD4 with 2CA, and the mixture was incubated for one hour. Then the native enzyme and that treated with 2CA were subjected to tryptic digestion. These peptidic fragments then were subjected to LC/MS/MS analysis to identify any 2CA modified peptide fragments in the drug treated samples, which were contrasted with that from native protein digestion. This fragment analysis led to the identification of the peptide, F850 LGEVHC*GTNVR (SEQ ID NO: 1). This peptide sequence corresponds to the active site region of PAD, and additionally Cys656 is the catalytic residue in the active site of PAD2 that is modified by an acetamidine moiety of 2CA confirming the covalent modification of PAD2 with 2CA.46 
MS Disease Attenuation In Vivo by 2CA in the ND4 Mouse Model.
The relevance of PAD inhibitors to preventing demyelination and potentially for the treatment of MS was investigated in mice using four independent models.46 The ND4 mouse is a transgenic mouse containing 70 copies of the cDNA for DM20 (a myelin proteolipid protein) which demyelinates spontaneously at 3 months of age. Heterozygous littermates are normal animals from birth until 10-12 weeks of age at which stage they spontaneously develop a non-autoimmune, primary progressive and ultimately fatal CNS demyelinating condition.49,50 In these mice, disease progression is associated with increased expression of PAD in myelin, and hypercitrullination of myelin protein and histone H3 proteins due to the enhanced levels of PAD.21,51 Thus, this is a good model to evaluate the effect of drugs on the demyelinating conditions such as MS.
For the in vivo efficacy determination, ND4 mice were administered 2CA (5 mg/kg) i.p. every other day. The drug treatment was initiated either well before disease onset at 2 months of age of the mice or during early stages after disease onset at 3.5 months of age, and mice were observed for a period of 4-5 months after the initiation of the treatment (FIG. 3). Early and prolonged 2CA administration essentially prevented the disease (triangle profile in FIG. 3A). Most untreated mice were sacrificed with severe disease by 6 months of age, while none died in the treatment groups and all mice in the treatment group received the treatment until the end of the study at 6 months. The second group of mice was administered 2CA after the early disease onset at age 3.5 months and they did not show disease progression during the treatment period of up to 6 months of age (FIG. 3B), but a mild disability continued. However, fully progressive clinical disease re-emerged promptly after therapy cessation at 6 months. The above data providing a temporal link between demyelinating disease protection by 2CA and relapse after therapy cessation place PAD-mediated citrullination and disease progression in the executive arm of transgene-driven pathogenesis in this demyelinating disease model.46 
Overall, it can be concluded from the above experiments that 2CA induced dramatic disease attenuation, but required continued treatment with the drug due to obvious persistence of pathogenic transgene expression.
In further analysis, it was observed that an untreated ND4 transgenic mouse brain exhibited citrullination levels (due to PAD2 and PAD4 activities) higher than those in a normal mouse brain (FIG. 4A, second bar from the left vs. leftmost bar). When PAD activity was observed right after the cessation of 2CA treatment (at 6 months), it was found to be attenuated and was almost equivalent to that observed in a normal mouse brain (FIG. 4A, third bar from the left). Two months after 2CA therapy cessation, however, PAD activity in the white matter of brain was observed to be considerably overshot (and rapid disease progression) (FIG. 4A, rightmost bar). In further analysis, PAD2 gene expression measured by its mRNA levels paralleled citrullination due to PAD activity, suggesting that disease-induced elevations in citrullination of MBP are regulated at the transcriptional level implying the participation of PAD2 (FIG. 4B).
The levels of PAD expression and the corresponding enzymatic activities, hypercitrullination and demyelination were further correlated with the morphological changes in myelin structure by transmission electron microscopy (TEM) of optic nerve cross-sections from the 6 months old mice right after 2CA treatment cessation (FIG. 5). In non-transgenic (normal) ND4 littermates, axons were well myelinated with myelin of uniform thickness (FIG. 5A, left panel). ND4 transgenic mice showed wide areas of myelin loss, and degradation and nude axons were common following development of the disease state after 3 months post-birth (FIG. 5A, middle panel). At 6 months of age, immediately following 2CA treatment, this morphology in ND4 mice was clearly improved, with few axons seriously affected (FIG. 5A, right panel). However, two months after the cessation of 2CA treatment, myelin loss and thinning of the axons reappeared (FIG. 5B). Luxol-fast-blue staining of myelin showed impressive myelin deficits and pronounced vacuolization in PBS-treated ND4 mice, defects which were dramatically improved in 2CA-treated mice.46 When treatment was ceased, myelinolysis re-emerged indicating disease progression, as indicated above.
To quantify the above myelin changes, G-ratios (axon diameter/fiber diameter) were calculated from ˜500 non-contiguous semi-thin sections per treatment group. Compared to healthy littermates (G-ratio 0.74+0.13), ND4 mice showed a reduction in optic nerve myelin thickness: G-ratio 0.96+0.3 (p=0.0013). In 2CA-treated ND4 mice, myelin thickness was slightly improved and showed less variation (G ratio: 0.9+0.15). These treatment data are typical for remyelination, where the original myelin thickness is never re-achieved. These results strongly suggest that 2CA, a PAD inhibitor, showed good efficacy in the ND4 transgenic mice attenuating the hypercitrullination-mediated demyelination, and promoting remyelination.
Disease Attenuation by 2CA in MOG-EAE Mouse Model.
A more commonly used fatal MOG-EAE model was also used to test the efficacy of 2CA and to understand the effects of PAD inhibitors on demyelination. Fatal EAE was induced in C57BL/6 mice with 100 μg of MOG35-55 peptide emulsified in Freund's complete adjuvant and 300 ng of pertussis toxin. At the earliest sign of disease, typically 9 days post-immunization, groups of mice received either PBS or 2CA (5 mg/kg i.p., every other day) (FIG. 6). Untreated mice developed progressive disease rapidly and were sacrificed when moribund around day 19 (FIG. 6B). When treated with 2CA starting day 9, treatment did not affect the disease course until day 14 (when compared to untreated mice, FIG. 6A). After day 14, disease progression halted, and recovery began, leaving ˜50% survival by day 30 (FIG. 6B)—a significant outcome in this aggressive model. When 2CA treatment was started before immunization, disease lethality was zero. Despite the severity of disease in this animal model, there was relatively little histopathology in brain. However, vacuolar demyelination and lymphoid infiltration were prominent in the spinal cord of the PBS-treated mice. In the 2CA treatment group, surviving mice showed much improved, virtually normal spinal histology (see ref. 46). Additionally, in a separate study using chronic relapsing EAE model, diseased mice showed significant improvement after receiving 2CA treatment (for details, see ref. 46).
In the 2CA-treated group, some scattered CD3+ T cells were still detected when very sensitive immunostaining was performed on samples from treated animals, but the heavy T cell dusters seen in PBS-treated controls were absent suggesting that when treated with 2CA, the resulting effect may be the suppression of tissue T cell expansion. PAD activities in the brain white matter of PBS-treated mice were elevated, as expected, and 2CA effectively attenuated this elevated PAD activity. In the spinal cord of the EAE animals also, PAD activities of PBS-treated group were 3-fold higher than that in the normal mice, but reductions to normal levels were once again observed following treatment with the PAD inhibitor 2CA. No relapse was observed in the 2CA-treated group.
Additional experiments using pMOG35-55 peptide, additional replacement peptides carrying one or two citrullines in place of one or two Arg residues in the offensive MOG peptide, indicated that the disease-related T cell autoreactivity repertoire prominently includes recognition of citruline-containing epitopes, an observation with precedence in the literature.52 This led to the conclusion that the inhibition of PAD activities by 2CA in the early phase of the EAE model produced a major reduction of autoreactive T cell pools.46 While not wishing to be limited by theory, this could provide a mechanistic explanation for the 2CA-induced failure to generate the massive T cell tissue invasion characteristic of the effector phase of this disease. The remaining infiltrates of scattered CD3+ T cells in treated survivors may be either anergic or non-specific bystanders with little pathogenicity, since there were no relapses after therapy cessation.
Overall, these in-depth studies to understand the effects of 2CA on the spontaneous demyelinating disease (ND4 transgenic mouse) model and the MOG-EAE model indicated a good potential for study of the inhibitors of deiminases to inhibit demyelination. 2CA has no specific structural features that provide specificity to PAD or its isozyme catalytic site. It is a polar molecule due to the acetamidine structure, as well as a reasonably reactive molecule (covalent inhibitor). It has the ability to react with a variety of nucleophiles in vivo causing irreversible modifications (FIG. 2).
Structures of PAD Enzymes.
Structurally, PAD enzymes are Ca2+-dependent enzymes that catalyze the conversion of arginine residues in proteins to citrulline via the deimination of the guanidinium moiety in the side chain of Arg residues.53,54 The structure consists of the N-terminal domain predominately folded into β-sheets, and the C-terminal domain where the catalytic site is located. The catalytic site, where the substrate binds, has two Asp residues, one His residue and a Cys residue that are involved in the deimination reaction. Acidic amino acids, Asp350 and Asp473, function as general base residues during the hydrolysis of the amine in the guanidinium moiety of the peptidyl arginines. These two Asp residues are located in the bottom of the substrate-binding pocket (FIG. 7). 2CA, due to its acetamidine structure carrying a positive charge, binds at this anionic pocket and modifies the Cys residue that is in close proximity (FIG. 2). 2CA does not carry any additional structural features that provide it with specificity to inhibit PAD enzymes only, and not any other similar enzymes.
Over the past decade, there have been only a handful of efforts focused on understanding various ligands, their interactions and the inhibitors targeting PAD enzymes, and most notably, various peptide derivatives to understand the substrate and inhibitor properties targeting PAD enzymes.39,55,56,57 The most potent non-peptidic compounds from these investigations are chlortetracycline, a tetracycline derivative with an IC50 of 100±10 μM as a competitive inhibitor and a substrate analog, F-amidine with an IC50 of 21±2.1 μM as an Irreversible inactivator.